Inflammation is a well-known mechanism associated with organic gastrointestinal (GI) disorders, such as the Inflammatory Bowel Disease. However, only recently has evidence emerged that an underlying inflammatory mechanism may be associated with the previously termed functional gastrointestinal disorders (FGID) associated with chronic GI symptoms such as diarrhea and chronic abdominal pain (CAP) with no identified organic cause. In our studies, we found evidence for differential levels of specific candidate biomarkers in GI biopsies from patients with histologically defined inflammatory phenotypes. The IL-6 levels in the lower GI biopsies from pediatric patients with inflammatory phenotypes were found to be significantly increased compared to control patients with non-inflammatory phenotypes (p=0.004). On the other hand, the number of mast cells were significantly higher in patients with no evidence of histological inflammation compared to patients with inflammatory phenotype (p=.049). Interestingly, no difference in IL-6 and mast cell staining was observed in the upper GI biopsies, hinting at the lower GI tract environment as a potential primary focus for investigation of relevant pathways underlying chronic GI symptoms (Henderson et al., 2012, WJGP). A noteworthy functional relevance of IL-6 from this finding is the multiple pathways that this molecule plays a role in. In addition to inflammation, IL-6 has been suggested by others to additionally contribute to the disruption of epithelial barrier integrity. These aforementioned findings provide credence for our ongoing prospective Brain-Gut Natural History protocol, which focuses on finding the systemic biological mechanisms contributing to aberrations in GI functions. Such aberrations in GI functions are manifested in the patients as chronic GI symptoms such as CAP and bowel movement changes, all of which significantly and functionally affect the quality of life of the patients. In this protocol, CAP is assessed by the Gastrointestinal Pain Pointer (GIPP), a newly invented technology (Henderson et al., licensing PST, Inc.) that has been developed, beta tested, and validated at the NIH Clinical Center. It electronically gathers data on the patients subjective assessments of the location, type, and intensity of GI symptoms, along with real-time synchronized objective measures of heart rate and blood pressure. The patients use a graphical interface to choose their gender and body type, record the location and intensity of the GI distress, and selects descriptive words for the GI discomfort. Along with GIPP, the short-form McGill Pain Questionnaire is also utilized for validation. The comparisons were conducted at interval time points of up to 5 hours following the patients ingestion of an intestinal permeability test solution, which induces GI discomfort. Time points were significantly correlated (p<.007) between the McGill and the GIPP. Thus, the GIPP is a valid and reliable instrument thereby allowing clinicians to have a more integrated resource for pain assessment that includes location, intensity, and quality along with physiologic parameters (Henderson, et al., CTS, 2012). In addition to the GIPP innovation, various laboratory tools and clinical measures are being employed either directly or indirectly in this protocol to study the potential mechanisms underlying the observed aberrations of GI symptoms. Specifically, the primary mechanisms of interest are the inflammatory pathways, the genetic changes in subclinical groups of patients, changes in stress-related outcomes and pathways, and changes in GI barrier function. Our results interestingly suggest that these mechanisms and pathways converge and influence one another at the molecular and cellular level. One of the clinical protocol measures employed is an intestinal permeability assessment, measured by urine analysis of four sugar probes absorbed at longitudinal tracts within the intestines. These probes are administered orally as a sugar solution to participants after an overnight fast. Excreted urine, injected with raffinose as an internal standard, was analyzed for individual sugar quantification via a novel mass spectrometry method. Based on these measurements, we recently showed that patients with CAP, meeting Rome III criteria for Irritable Bowel Syndrome (IBS), had a significantly decreased colonic permeability compared to control patients with no CAP (Del Valle-Pinero, et al., 2013). This phenomenon was represented by differential sucralose urine output in CAP patients versus control patients (p=0.011). Of the CAP patients, 55% had diarrhea. Changes in sucrose urine output, representing gastric permeability, and changes in lactulose-to-mannitol ratio, representing small intestinal permeability, were not observed (Del Valle-Pinero, et al., 2013). This unexpected finding, which points to altered colonic permeability as one of the likely mechanisms for the observed GI aberrations in patient with chronic GI symptoms, together with our histological evidence from the lower GI biopsies, serve as a starting point for a closer investigation into candidate cellular and molecular players within the pathways and microenvironment contributing to chronic GI symptoms. Genetic profiling provides further clues to relevant molecular pathways leading to the GI symptoms manifested in our patient cohort. We found an upregulation of the gene coding the pro-inflammatory cytokine IL-1a; which suggests that the mechanism behind stress-related GI symptoms is pro-inflammatory in nature. In another experiment, we compared IBS patients with a history of CAP to age-matched healthy controls via quantitative PCR expression of 96 inflammatory genes. Significant upregulation (7.46-fold) of the chemokine C-C motif ligand 16 (CCL-16) gene expression was found in IBS patients compared to controls. Within subsets of IBS patients, the CCL-16 gene expression was also upregulated in IBS-C patients, both compared to control (199.78-fold) and compared to IBS-D patients (138.47-fold; Del Valle Pinero, et al., 2011). In light of the recent finding by others elucidating enhancement of CCL-16 protein function as a haptotactic gradient regulator for two key inflammatory cells, the monocytes and the lymphocytes, by matrix metalloproteases, our finding provides further evidence for an inflammatory mechanism in these patients. More importantly, our finding offers a valuable insight of the molecular pathways which likely occur in the GI microenvironment under chronic GI distress. Further genetic clues of the key molecular players are provided by investigation of mRNA as well as microRNA levels. Our analysis of microRNA (miR) profiles in IBS patients reveals the downregulation of two oncogenic miRs, miR-342-3p and miR-150, compared to control patients. The involvement of inflammation is again highlighted by these specific miRs differential profiles in the patients. Finally, it has been increasingly recognized by others that the human microbiome compositions play a potentially crucial role in the pathogeneses of many disorders, as well as in pain and behavioral pathways. Our current studies aim to elucidate the composition of microbial communities at specific sites along the GI tracts in patients with and without CAP. Collectively, these data will be analyzed not only in reference to the primary clinical condition, but also in the context of specific demographic, anthropometric, plethysmographic, and psychological condition of the patients. The revealed mechanisms with associated biomarkers are anticipated to aid in the design of personalized biobehavioral interventions to improve clinical outcomes.